Methods of equalizing representation levels of nucleic acid targets

ABSTRACT

The disclosure provides methods of reducing the range of representation levels of nucleic acid targets. The methods are particularly useful for multi-target analyses benefiting from a low variance of target representations, such as, e.g., single molecule sequencing and/or heterozygous genotyping, and pathogen diagnosis. Two general methods are provided. In Method 1, starting concentrations of probes are adjusted. In Method 2, target-specific probes are “binned,” i.e., several subsets of probes are selected based on similar representation levels. Thereafter, each subset of corresponding targets is extracted, with or without amplification, using a separate portion of the sample (i.e., separate vessels).

TECHNICAL FIELD

The invention is in the field of molecular biology and relates tomethods for nucleic acid analysis. In particular, the invention relatesto methods for equalizing nucleic acid target representation yields inprobe-specific extraction of targets.

BACKGROUND

Many types of studies require analysis of a large number of genomicregions, with as many as 200,000 target regions analyzed. Normally, eachtarget region is amplified in a separate reaction in an individualsample. The sample requirements and expense of so many separatereactions makes studies with large numbers of genomic targetsprohibitive. The complete analysis of complex multi-component systems isfrequently beyond the capability of existing methods. Simplification ofthe system, i.e., selection of the key components and discarding others,allows a useful study to be designed.

In the case of nucleic acid sequencing, isolation of the target sequenceis frequently realized by amplification (e.g., by PCR). One solution isto combine the multiple amplification and isolation reactions into asingle vessel, i.e., multiplexing. However, two problems arise. First,for PCR, the interaction between the probes generates large numbers ofspurious products if multiple probes are combined. Even if this problemis solved, the efficiency of isolation is not uniform, resulting in alarge range of concentrations for the selected targets. Thisconcentration range then requires a large dynamic range of sensitivityfor the subsequent analysis. For example, a typical DNA chip has auseful sensitivity range of ˜30×. If the PCR amplicon mixture is probedwith such a DNA chip, all amplicons below the 30× range could not bedetected. Targeted gene sequencing presents a similar problem. A singlegene is about 1 part in 10⁵ of a whole human genome, while the exons inone gene are about 2 parts in 10⁶ of a whole genome. Thus, a typicaltargeted gene study involving 10-500 genes would require the extractionof 100-5000 separate gene fragments per sample. Furthermore, a genomicsample of 3 μg of human DNA contains about 10⁶ copies of the genome, andthe range of isolated genes would vary in copy number by 100-1000×.Additionally, some applications, such as heterozygous genotyping, mayrequire read depths of 20× or more, which translates the whole samplebeing sequenced to a depth of ˜20,000×, making the cost of the data veryhigh. If all fragments could be extracted in one or a few reactions(i.e., multiplexed) much less sample would be required, resulting inlower reagent consumption and labor costs. However, none of theavailable multiplexing methods provide uniform efficiency.

Accordingly, there is a need for methods for isolation of multiple geneswith substantially similar representation levels.

SUMMARY OF THE INVENTION

Simultaneous probe-specific extraction of multiple nucleic acid targetstypically yields a wide range of target representations in the finalsample. Multiple factors contribute to this variance, both at the targetcapture stage as well as during amplification of targets. The inventionprovides methods of equalizing the variation in nucleic acid targetrepresentations in the final sample containing the extracted targets(amplified or non-amplified). Using methods of the invention, thetargets' range of representation levels for a given set ofprobes/targets is generally reduced relative to the range obtainedotherwise. The methods described here are particularly useful for thosetypes of multi-target analysis that benefit from lower variance oftarget representations, such as, for example, single moleculesequencing, heterozygous genotyping, and pathogen diagnostics.

An initial range of representation levels is first determined for agiven set of probes/targets. To that end, a plurality of selectiveprobes is contacted with a sample containing targets, and allprobe-specific targets are extracted together. In some embodiments, amultiplex amplification of targets is also performed. These extracted,and optionally amplified, targets form the initial range ofrepresentation levels.

The invention provides two general methods (Method 1 and Method 2) thatreduce the target representation variance in the analytic sample.

In Method 1, the starting concentrations of probes are adjusted up ordown based on their initial representation levels. For example, thestarting concentrations of the probes yielding lower representationlevels are increased, while the starting concentrations of the probesyielding higher representation levels are reduced.

In Method 2, the probes are “binned,” i.e., several subsets of probesare selected based on similar representation levels. For example, probeswith low, medium, and high representation levels may be grouped,respectively, into three subsets. Thereafter, each subset ofcorresponding targets is extracted, with or without amplification, usinga separate portion of the sample (i.e., separate vessels). Forsubsequent analysis, the resulting targets may be combined into a singlesample in proportions adjusted so that a reduced range is maintained.For example, a larger portion of the low-representation subset iscombined with a smaller portion of high-representation subset. In someembodiments of Method 2, multiplex amplification of targets with binnedprobes is performed in separate portions of the sample, thereupon, theseparate reactions are combined.

In some embodiments, the targets are captured by circularizing probesannealed to their respective targets and removing non-circularizednucleic acids prior to the target isolation.

In some embodiments, targets are analyzed by sequencing, e.g.,sequencing by synthesis. In preferred embodiments, the sequencing isperformed at a single molecule resolution, e.g., with the targetsimmobilized on a support.

Additional aspects of the invention are described in detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides results of the study described in the Example. Thediagram shows the number of amplicons detected vs. probe identity asnumbered from 1 to 518.

FIG. 2 provides results of the study described in the Example. The graphshows the relative abundance of the extracted targets.

FIG. 3 provides calculated sequencing depth required to “cover” theamplicon pool used in the Example.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for reducing the range of representationlevels of nucleic acid targets in biological samples. According to themethods, the range of target representation levels for a given set oftargets/probes is reduced, generally, relative to that obtained withoutsuch methods. The targets may be captured and analyzed directly, or theymay be amplified, for example, by multiplex PCR amplification(amplification of two or more target sequences in the same reaction).

In general, an initial range of representation levels is firstdetermined for a given set of probes/targets. To that end, a pluralityof selective probes is contacted with a sample containing targets, andall probe-specific targets are extracted together. In some embodiments,a multiplex amplification of targets is performed. These extracted andoptionally amplified targets form the initial range of representationlevels.

The range of representation levels typically is either the difference orthe ratio of the two parameters: 1) the abundance of the mostrepresented target and 2) the copy number of the least representedtarget in the sample. (Outliers are generally excluded, but need not bein some implementations.) The abundance of a target may be determined orexpressed indirectly as C_(t), mass, copy number, a number or thepercentage of sample fractions containing a target, etc. Therepresentation range can be determined by any suitable method. Theinitial representation range will vary depending on the nature of thebiological sample, the specificity of the probes, the initialconcentration of each target in the sample, the initial concentration ofthe probes, efficiency of the amplification process and other factors.In some embodiments, the initial range of representation levels is atleast 100×, for example, 200×, 500×, 1,000×, 10,000× or higher.

The invention provides at least two methods (generally referred to as“Method 1” and “Method 2”) to reduce target representation variance inthe final sample. The methods can be used individually or in combinationwith each other.

Method 1—In Method 1, starting concentrations of probes are adjusted.The starting concentrations of the probes yielding lower representationlevels are increased, while the starting concentrations of the probesyielding higher representation levels are reduced. Generally, Method 1comprises the following steps:

-   -   a) contacting a plurality of probes with a sample comprising a        plurality of targets;    -   b) capturing the plurality of nucleic acid targets with the        plurality of probes;    -   c) optionally, amplifying the captured targets using the        plurality of probes;    -   d) determining an initial range of representation levels of the        captured nucleic acid targets;    -   e) repeating steps a), b), and c) (if the latter is performed),        at starting concentrations of the probes adjusted to reduce the        range of representation levels of captured targets relative to        the initial range, thereby producing a final sample.

In some embodiments, the starting concentrations of at least 5, 10, 50,100 probes are adjusted at least 2-, 3-, 4-, 5-, 10-fold or more up ordown. In some embodiments, the probes are divided into 2, 3, 4, 5, 10,or more subsets according to the similarity of their representationlevels, and the starting concentration of the entire subset is thusadjusted.

Method 2—In Method 2, the probes are “binned,” i.e., several subsets ofprobes are selected based on similar representation levels. Then, eachsubset of corresponding targets are extracted individually (with orwithout amplification) using a separate portion of the sample (theprocedure generally referred to as “binning”). The resulting isolatedtargets may then be combined into a single sample at various proportionsso as to maintain a reduced range of target representation levels.Generally, Method 2 comprises the following steps:

-   -   a) contacting a plurality of probes with a sample comprising a        plurality of targets;    -   b) capturing the plurality of nucleic acid targets with the        plurality of probes;    -   c) optionally, amplifying the captured targets using the        plurality of probes;    -   d) determining an initial range of representation levels of the        captured nucleic acid targets;    -   e) selecting two or more subsets of targets/probes so that each        of the subsets contains targets of a range of representation        levels which is reduced relative to the initial range; and    -   f) repeating steps a), b), and c) (if the latter is performed),        using separate samples so that each sample contains only        targets/probes of different selected subsets.

In some embodiments, the Method 2 further comprises:

-   -   g) combining the samples produced in step f) or portions thereof        to produce a final sample.

In both Method 1 and Method 2, the range of representation is reduced inthe final sample relative to the initial range. In some embodiments, thereduced range in the final sample (e.g., before analysis) is less than50×, 30×, 20×, 10×, 5×, 3×, 2× or lower. Accordingly, in someembodiments, the range of representation levels is reduced by at least2-, 5-, 10-, 50-, 100-, 500-, 1.000-fold or more.

Multiplex amplification—In some embodiments, the targets are amplifiedin a multiplex fashion prior to analysis. Target sequences are typicallyamplified using respective sequence-specific probes. As many as hundredsor a greater number of targets may be amplified in the same reaction.Most commonly, multiplex amplification of nucleic acids is performedusing the polymerase chain reaction (PCR), however, the invention mayalso be used with other amplification techniques amenable tomultiplexing, including the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the nucleic acidsequence-based amplification (NASBA), the strand displacementamplification (SDA), rolling circle amplification (RCA), hyper-branchedRCA (HRCA), etc. Numerous target-specific probes are available fromcommercial sources. A desired set of probes can also be syntheticallymade using conventional nucleic acid synthesis techniques. For example,probes may be synthesized on an automated DNA synthesizer using standardchemistries, such as, e.g., phosphoramidite chemistry. In the methods ofthe invention, a plurality of target-specific probes may comprise two ormore probes specific to different targets, e.g., 3, 4, 5, 10, 50, 100,200, 300, 500, 1,000, 10,000 or more. All or some of the probes willhave corresponding targets in a given sample. Thus, a multiplexamplification reaction may be performed on two or more targets, e.g., 3,4, 5, 10, 50, 100, 200, 300, 500, 1,000, 10,000 or more.

Circularization—In some embodiments of Methods 1 and 2, the methodfurther includes circularizing probes annealed to respective targets.Following the circularization of the target, the linear (non-target)nucleic acids are removed from the sample. Next, the circularizedtarget/probe is linearized and the target is bound to a support forfurther analysis. Circular constructs for PCR-based amplification havebeen previously described (see, e.g., PCT Application Publication No. WO2005/111236). Circularization of nucleic acids has been previously usedto increase efficiency of PCR-based amplification of nucleic acids (see,e.g., Dahl et al. (2005) Nucleic Acid Res., 33, e71; and Dahl et al.(2007) Proc. Natl. Acad. Sci., 104:9387-9392). Circularization oftargets/probes in the context of sequencing is described U.S. patentapplication Ser. No. 11/958,173. In general, a probe used forcircularization comprises: 1) a double-stranded nucleic acid having twooverhang ends that are specific (i.e., complementary) to two sites ofthe target nucleic acid, 2) one or more cleavage site(s) in thedouble-stranded region of the probe, and 3) other optional elements.Accordingly, prior to the amplification, Methods 1 or 2 may includeligating the probes and the targets to form closed circular nucleicacids and removing non-circularized linear nucleic acids (e.g., byexonuclease treatment).

Nucleic Acid Targets

Target nucleic acids may come from a variety of sources. For example,nucleic acids may be naturally occurring DNA or RNA (e.g., mRNA ornon-coding RNA) isolated from any source, recombinant molecules, cDNA,or synthetic analogs. For example, the target nucleic acid may includewhole genes, gene fragments, exons, introns, regulatory elements (suchas promoters, enhancers, initiation and termination regions, expressionregulatory factors, expression controls, and other control regions), DNAcomprising one or more single-nucleotide polymorphisms (SNPs), allelicvariants, and other mutations. The target nucleic acid may also be tRNA,rRNA, ribozymes, splice variants, antisense RNA, or siRNA.

Target nucleic acids may be obtained from whole organisms, organs,tissues, or cells from different stages of development, differentiation,or disease state, and from different species (human and non-human,including bacteria and virus). Various methods for extraction of nucleicacids from biological samples are known (see, e.g., Nucleic AcidsIsolation Methods, Bowein (ed.), American Scientific Publishers, 2002).Typically, genomic DNA is obtained from nuclear extracts that aresubjected to mechanical shearing to generate random long fragments. Forexample, genomic DNA may be extracted from tissue or cells using aQiagen DNeasy Blood & Tissue Kit following the manufacturer's protocols.

The length of the target nucleic acid may vary. The average length ofthe target nucleic acid may be, for example, at least 300, 350, 400,450, 500, 550, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 nts orlonger. In some embodiments, the length of the target is between 300 and5000 nts, 400 and 4000 nts, or 500 and 3000 nts.

Analysis Methods, Including Sequencing

In both Method 1 and Method 2, the final sample may be subjected toadditional manipulations, and if desired, analyzed by any suitablemethod, e.g., sequencing, for example, for exonic re-sequencing,genotyping, single nucleotide polymorphism (SNP) detection) or used forallele quantification, pathogen diagnostics, etc. Methods of theinvention are particularly useful for those types of nucleic acidanalysis that benefit from a low variance of target representations. Theinvention can be used with any suitable analysis method, includingsequencing, detection with hybridization probes, etc.

In preferred embodiments, the nucleic acid targets are analyzed bysequencing by synthesis. For example, in single-molecule sequencing, itmay be advantageous to reduce the variance so that the requiredsequencing depth (also referred to as “sequence coverage”) is reduced.This requirement generally is a function of the importance of achievinga high probability that each base in a sequence will be measured, andmeasured accurately within acceptable error tolerances. Errortolerances, and even the need to measure each base, may vary dependingon the purpose of the experiment. Accordingly, in some embodiments, thesequencing applications include sequencing by synthesis with sequencecoverage being above 1×, 2×, 3×, 5×, 10×, 20× or higher. For example,for a given set of targets with a total length of 1,000,000 bases, 1×coverage means that 1,000,000 bases are sequenced, some of which may bemultiples of the same sequence.

Any suitable sequencing method may be used, including Sanger sequencing.For example, the following sequencing platforms are currently available:the Genome Sequencers from Roche/454 Life Sciences, the 1G Analyzer fromIllumina/Solexa, and the Heliscope system from Helicos Biosciences. Twosequencing-by-ligation platforms are also currently available: the SOLiDsystem from Applied BioSystems and the Polonator G.007 from DanaherMotion. Additional sequencing platforms have also been described byPacific BioSciences and VisiGen Biotechnologies. Each of these platformscan be used in the methods of the invention. In some embodiments, thesequencing platforms used in the methods of the present invention haveone or more of the following features:

-   -   1) four differently optically labeled nucleotides are utilized        (e.g., 1G Analyzer, Pacific BioSciences, and Visigen);    -   2) sequencing by ligation is utilized (e.g., SOLiD, Polonator);    -   3) pyrophosphate detection is utilized (e.g., Roche/454);    -   4) four identically optically labeled nucleotides are utilized        (e.g., Helicos);    -   5) fluorescent energy transfer (FRET) is utilized (e.g.,        Visigen).

In some embodiments, a plurality of nucleic acid molecules beingsequenced is bound to a support. To immobilize the nucleic acid on asupport, a capture sequence/universal priming site can be added at the3′ and/or 5′ end of the template. The nucleic acids may be bound to thesolid support by hybridizing the capture sequence to a complementarysequence covalently attached to the solid support. The capture sequence(also referred to as a universal capture sequence) is a nucleic acidsequence complementary to a sequence attached to a solid support thatmay dually serve as a universal primer. In some embodiments, the capturesequence is polyN_(n), wherein N is U, A, T, G, or C, n≧5, e.g., 20-70,40-60, e.g., about 50. For example, the capture sequence could bepolyT₄₀₋₅₀ or its complement.

As an alternative to a capture sequence, a member of a coupling pair(such as, e.g., antibody/antigen, receptor/ligand, or the avidin-biotinpair as described in, e.g., US Pat. App. Pub. No. 2006/0252077) may belinked to each fragment to be captured on a surface coated with arespective second member of that coupling pair.

The solid support may be, for example, a glass surface such as describedin, e.g., US Pat. App. Pub. No. 2007/0070349. The surface may be coatedwith an epoxide, polyelectrolyte multilayer, or other coating suitableto bind nucleic acids. In preferred embodiments, the surface is coatedwith epoxide and a complement of the capture sequence is attached via anamine linkage. The surface may be derivatized with avidin orstreptavidin, which can be used to attach to a biotin-bearing targetnucleic acid. Alternatively, other coupling pairs, such asantigen/antibody or receptor/ligand pairs, may be used. The surface maybe passivated in order to reduce background. Passivation of the epoxidesurface can be accomplished by exposing the surface to a molecule thatattaches to the open epoxide ring, e.g., amines, phosphates, anddetergents.

Subsequent to the capture, the sequence may be analyzed, for example, bysingle molecule detection/sequencing, e.g., as described in the Exampleand in U.S. Pat. No. 7,283,337, including template-dependentsequencing-by-synthesis. In sequencing-by-synthesis, the surface-boundmolecule is exposed to a plurality of labeled nucleotide triphosphatesin the presence of polymerase. The sequence of the template isdetermined by the order of labeled nucleotides incorporated into the 3′end of the growing chain. This can be done in real time or can be donein a step-and-repeat mode. For real-time analysis, different opticallabels to each nucleotide may be incorporated and multiple lasers may beutilized for stimulation of incorporated nucleotides.

The following Example provides illustrative embodiments of the inventionand does not in any way limit the invention.

Example Equalization of a 518-Probe Set

Tags are prepared as described through step C in FIG. 1 of Dahl et al.(2007) PNAS, 104:938, from a pool of 518 probes, except that the probeconcentrations were adjusted up or down up to 10× from the formerconcentration according to the relative representation found in Dahl.The sample was then a multiplex pool of 518 PCR amplicons with variablerepresentation. The entire probe pool is then subjected to a polyAtailing process substantially as described in US Pat. App. Pub. No.2007/0070349. On average, 25 A bases were added to each amplicon in thepool followed by a ddA-Cy3 to terminate and label the amplicon. ThesepolyA-tailed amplicons were then captured onto a sequencing surfacespecially prepared for this test. The surface bound capture consisted ofthe PCR primer complement and by dT20 with the dA20 nearest the surface.In this way, 38 bases of single-stranded sequence, complementary to the3′ end of each amplicon, was available on the surface to both capturethe amplicons by hybridization and act as a sequencing-by-synthesisprimer. Since all the amplicons are still whole, only the first 20-30bases will be read. However, this is more than sufficient to “count”representation of the extracted tags. The sample is sequenced, one passsequencing up substantially as described in US Pat. App. Pub. No.2007/0070349. The sequences are aligned against a dictionary of words 50bases long derived from the known target sequences of the probes. Thefirst 20 bases of each word are the same as the probe sequences. In thisway, a count of each probe can be made.

FIGS. 1-3 provides results of a representative study performedsubstantially as described above. The plot in FIG. 1 is the number ofamplicons detected vs. probes identity as numbered from 1 to 518. Thereis no significance to the order of the probes. Results are shown in FIG.2 expressed as relative abundance of extracted targets. 89,000 targetprobes were found. Plotted is the abundance of the 518 targets relativeto the most abundant, normalized to 1.0 for this most abundant target.For example, targets plotted at 0.01 are 1% as abundant as the mostabundant target. In an ideal process, this plot would be a nearlyvertical line, all probes present at the same number.

A calculated sequencing depth required to “cover” the amplicon pool isrepresented in FIG. 3. The calculation was performed as follows: 1)obtain the total length of all amplicons (˜110,000 bases); 2) for eachof the 110,000 bases sequenced, calculate probability of havingsequenced an amplicon. For the first 110,000 bases (referred to as 1×coverage), the statistical estimate is to have sequenced 36% of theamplicons (of note, some of the amplicons at the 36% most abundant probewill be missed and some less abundant amplicons will be seen). For550,000 bases sequence (5× coverage), one would expect to see at least 1copy of 90% of the amplicon pool. For 1.1 million bases sequenced (10×coverage), one would expect to see at least 1 copy of 96% of theamplicons present. In practice, the 10× coverage is typicallysufficient. A 50× coverage would provide at least 1 copy of 99% of theamplicons.

All publications, patents, patent applications, and biological sequencescited in this disclosure are incorporated by reference in theirentirety.

1. A method of equalizing representation levels of nucleic acid targets,the method comprising: a) contacting a plurality of probes with a samplecomprising a plurality of targets; b) capturing the plurality of nucleicacid targets with the plurality of probes; c) optionally, amplifying thecaptured targets using the plurality of probes; d) determining aninitial range of representation levels of the captured nucleic acidtargets; and e) repeating steps a), b), and c) if performed, at startingconcentrations of the probes adjusted to reduce the range ofrepresentation levels of captured targets relative to the initial range,thereby producing a final sample.
 2. The method of claim 1, wherein thestep of amplifying the captured targets is required.
 3. The method ofclaim 1, wherein the targets are amplified by multiplex PCR.
 4. Themethod of claim 1, wherein the methods further comprises f) analyzingthe targets obtained in the final sample.
 5. The method of claim 4,wherein the targets are analyzed by sequencing.
 6. The method of claim5, wherein the sequencing is sequencing by synthesis or ligation.
 7. Themethod of claim 6, wherein the sequencing by synthesis is performed at asingle molecule resolution.
 8. The method of claim 1, wherein thecapturing step comprises circularizing probes annealed to targets andremoving non-circularized nucleic acids.
 9. The method of claim 1,wherein the range of representation levels is reduced by at leasttwo-fold.
 10. The method of claim 1, wherein the initial range ofrepresentation levels is greater than 100× and/or the reduced range ofrepresentation levels is less than 50×.
 11. The method of claim 1,wherein the starting concentrations of at least five probes are adjustedat least two-fold up or down.
 12. A method equalizing representationlevels of nucleic acid targets, the method comprising: a) contacting aplurality of probes with a sample comprising a plurality of targets; b)capturing the plurality of nucleic acid targets with the plurality ofprobes; c) optionally, amplifying the captured targets using theplurality of probes; d) determining an initial range of representationlevels of the captured nucleic acid targets; e) selecting two or moresubsets of targets/probes so that each of the subsets contains targetsof a range of representation levels which is reduced relative to theinitial range; and f) repeating steps a), b), and c) if performed, usingseparate samples so that each sample contains only targets/probes ofdifferent selected subsets.
 13. The method of claim 12, wherein themethod further comprises g) combining the samples produced in step f) orportions thereof to produce a final sample.
 14. The method of claim 12,wherein the step of amplifying the captured targets is required.
 15. Themethod of claim 14, wherein the targets are amplified by multiplex PCR.16. The method of claim 15, wherein in two or more multiplexamplification reactions at least five targets are amplified in each. 17.The method of claim 12, further comprising h) analyzing targets in thecombined sample.
 18. The method of claim 17, wherein the targets areanalyzed by sequencing.
 19. The method of claim 18, wherein thesequencing is sequencing by synthesis or ligation.
 20. The method ofclaim 19, wherein the sequencing by synthesis is performed at a singlemolecule resolution.
 21. The method of claim 12, wherein the capturingstep comprises circularizing probes annealed to respective targets andremoving non-circularized nucleic acids.
 22. The method of claim 12,wherein the range of representation levels is reduced by at leasttwo-fold.
 23. The method of claim 12, wherein the initial range ofrepresentation levels is greater than 100× and/or the reduced range ofrepresentation levels is less than 50×.
 24. The method of claim 12,wherein five or more subsets of probes are selected.
 25. The method ofclaim 1, wherein two or more subsets of probes comprise at least fiveprobes each.